Structure determination of membrane proteins is an important challenge for biomedical science. About thirty percent of expressed proteins span lipid bilayers, yet structures of only about one hundred membrane proteins have been resolved. Membrane proteins are encoded by 20-35% of genes but represent fewer than one percent of known protein structures to date. Knowledge of their structures will be enormously insightful for cell biology. Furthermore, membrane proteins are important as drug targets. The slow rate of membrane-protein structure determination represents a significant bottleneck for both basic and applied bioscience discovery. This bottleneck largely derives from difficulties in forming well-ordered three-dimensional crystals of membrane proteins. Solution NMR presents an attractive alternative for the study of membrane proteins, as high-resolution structural information can be obtained for proteins up to 80 kD in size without the need for crystallization. Residual dipolar couplings (RDC's), commonly measured for biological macromolecules weakly aligned by liquid-crystalline media, are important global angular restraints for NMR structure determination. For membrane proteins greater than 15-kDa in size, Nuclear-Overhauser-effect (NOE)-derived distance restraints are difficult to obtain, and RDC's could serve as the main reliable source of NMR structural information. In many of these cases, RDC's would enable full structure determination that otherwise would be impossible. However, none of the existing liquid-crystalline media used to align water-soluble proteins are compatible with the detergents required to solubilize membrane proteins.
For solution NMR, macromolecules must be solubilized in water to facilitate fast tumbling; the faster the tumbling, the better the spectra. To promote water solubility, membrane proteins must be complexed with detergent micelles. The micelle-protein complex is considerably larger than the protein alone, and tumbling is relatively slow as a result. This increase in effective size is especially problematic for α-helical membrane proteins greater than 15 kD in size, where resonance peaks are closely spaced and become irresolvable with the fast coherence relaxation of slowly tumbling macromolecules. In order to obtain information about the internuclear angles, each protein must be made to tumble in a weakly ordered regime. The appropriate weak ordering, about 0.1%, can be achieved by dissolving the protein in an appropriate concentration of a suitable alignment material. For example, water-soluble proteins can be aligned weakly by a suitable amount with ˜1.5-2% Pfl filamentous phage, which forms a liquid crystal at that concentration. The easiest method for weak alignment of proteins is through mixing the protein with a liquid-crystalline medium, such as Pfl filamentous phage, DMPC/DHPC bicelles, C12E5 polyethylene glycol, or cellulose crystallites. However, none of these media are compatible with detergent-solubilized membrane proteins.
The general applicability of solution NMR spectroscopy to structural characterization of intact α-helical membrane proteins has been demonstrated by the structure determination of the 15-kDa Mistic protein and the 30-kDa pentameric phospholamban, as well as the complete assignment of backbone resonances and secondary structures of the 44-kDa trimeric diacylglycerol kinase and the 68-kDa tetrameric KcsA potassium channel. Despite such progress, full-scale structure determination of α-helical membrane proteins remains challenging and rare. Due to the large fraction of methyl-bearing residues in membrane proteins and to the added molecular weight of detergent micelles, the low chemical-shift dispersion of α-helical proteins is obscured by resonance overlap and line broadening, making assignment of side-chain methyl resonances extremely difficult. Without side-chain chemical shifts, it is impossible to obtain a sufficient number of long-range NOE-derived distance restraints for folding secondary segments into the correct tertiary structure. Therefore, development of alignment media for accurate RDC measurements from α-helical membrane proteins would enhance significantly the capability of solution NMR in structure determination of this important class of targets.
The most effective method for weak alignment involves mixing the protein of interest with large particles that form stable liquid crystals at low concentration (˜1.5-5% w/v). Liquid crystals that have been used to align water-soluble proteins include DMPC/DHPC-bicelle liquid crystals, filamentous phage particles, ternary mixtures of cetylpyridinium Cl/Br, hexanol, and sodium Cl/Br, binary mixtures of polyethylene glycol and hexanol, and cellulose crystallites. However, none could be applied to membrane proteins due to incompatibility with the zwitterionic or anionic detergents typically used to solubilize membrane proteins for structural study. The only method currently available for weak alignment of membrane proteins involves the use of strained (radially or axially compressed) polyacrylamide gels. However, dissolving protein-micelle complexes to high concentration in gels is notoriously difficult due to the inhomogeneous pore size of randomly cross-linked gel matrices. Thus the measured RDC's are of limited accuracy.
Nucleic acid nanotube liquid crystals can extend the advantages of weak alignment to NMR structure determination of a broad range of detergent-solubilized membrane proteins. Alignment media comprised of 800 nm heterodimer DNA nanotubes should be broadly useful for providing global structural restraints in solution NMR studies of membrane proteins. As a large number of helical membrane proteins of great biomedical interest are between 20-30 kDa in size—well below the current size limitation of solution NMR spectroscopy—new experimental systems for obtaining NMR structural information in the presence of detergents are of fundamental importance. DNA nanotechnology, which affords versatile molecular design and sub-nanometer-scale precision, has been pursued as a route towards building host lattices to position guest macromolecules for crystallographic structural studies. The present invention employs solution NMR instead of crystallographic methods, and validates the potential of DNA nanotechnology for imposing order on target macromolecules to acquire atomic-resolution structural information.